Cell intrinsic mechanisms exist to regulate hematopoietic stem cell (HSC) self-renewal. Self-renewal ability confined to the normal stem cells is essential for maintaining tissue integrity and is responsible for regeneration after injury. When acquired by aberrant cells, however, it could produce a self-renewing cancer stem cell that is refractory to treatments. Understanding the mechanisms of self-renewal is important for therapeutic expansion of HSCs as well as for eradicating the malignant roots of blood cancers. Contrasting to mRNA expression, microRNA expression faithfully marks developmental history and/or stage. HSCs develop through a well characterized step-wise differentiation process with a gradual loss of the self- renewing capacity. Disruption of the microRNA biogenesis key enzyme, Dicer, completely ablated stem cell activity. We thus hypothesized that specific microRNAs are required in regulating these key steps and the expression of some microRNAs may correlate with the ability to self-renew. Two complementary approaches were used to identify such specific microRNAs. By scanning the 3'UTR regions of genes known to be important for hematopoiesis, we identified that miR-150 exerted its function by targeting c-Myb. Additionally, microRNA expression profiling was performed using primary stem cells and their non-renewing immediate descendant cells. Based on the microRNA expression profile, we identified the miR-99b-let-7e-miR-125a cluster which dramatically expanded the hematopoietic compartment after long term transplantation in vivo. propose to gain detailed understanding of these microRNA species in regulating HSC self-renewal through two specific aims. 1. Determine the role of miR-150 in HSC function using miR-150 knockout mice. 2. Define the role of the miR-99b-let-7e-125a cluster in enhancing HSC self-renewal and assess its candidate molecular targets. These aims will be achieved through the combinational use of genetically engineered mouse strains and the appropriate in vivo and in vitro assays readily available in Dr. Scadden's laboratory. RELEVANCE (Seeinstructions): The proposed study promises the discovery of microRNA regulators for HSC self-renewal. Such microRNAs will provide new paradigms for HSC regulation. They will also serve as research tools to uncover additional molecular pathways and networks that confer the self-renewal phenotype, not only to normal HSCs, but also to leukemia stem cells.